Each organism is required, in the course of growth to synthesize new cell substances. There are numerous cell components, like, for example, amino acids and porphyrins which are to be newly formed starting from metabolites of the citrate cycle. This means that the metabolites involved in the citrate cycle must be newly synthesized. In the growth of microorganisms on acetate, ethanol or fatty acids, metabolites of the acetate cycle are newly synthesized by a reaction sequence which has been called the glyoxylate cycle (Kornberg, Biochemical Journal 99 (1966) 1-11), key enzymes for the glyoxylate cycle being the enzymes isocitrate lyase and malate synthase. Since the named enzymes in many organisms can grow exclusively on acetate, ethanol or fatty acids, but not on carbohydrates, the activity or the new synthesis of the two enzymes is mainly regulated by the carbon source of the medium.
Because of their club-like configuration, Corynebacterium glutamicum and the closely associated C. melassocolae, B. flavum and B. lactofermentum are counted as coryneform bacteria. These types of bacteria also belong to the known class of "glutamic acid bacteria" since they are capable under certain growth conditions of liberating large amounts of glutamate in the medium. The named microorganisms are of considerable industrial interest since they can be used for the production of amino acids, purines and proteins. For C. glutamicum, C. melassecolae, B. flavum and B. lactofermentum, growth upon acetate or ethanol is already known and it has been found that they are involved in a glyoxylate cycle, i.e. also utilize the enzymes isocitrate lyase and malate synthase (for an overview see Kinoshita, Amino acids, in Biology of Industrial Organisms, 1985, pages 115-142, Benjamin/Cummings Publishing).
In spite of long term industrial use of these organisms only recently have molecular biological methods been developed with the aid of which coryneform bacteria can be genetically modified for certain specific purposes. As a rule, the gene to be cloned is cloned under the control of its own promoters on vectors which are available in higher copy numbers in coryneform bacteria. It has been found in many cases that a strong overexpression of individual genes is a drawback to the growth of coryneform bacteria and thus has an effect on the production of desired products. This has its origin in an overproduction of the corresponding gene products to toxic effects within the metabolism of the cell and gives rise to a reduction in the growth of these cell. An example of such a case is the homologous overexpression of mutated genes which code for deregulated enzymes, i.e. such enzymes whose activity no longer has end product blocking, for instance, the homologous overexpression of the HOM 1 gene which codes for a deregulated homoserine dehydrogenase (Reinscheid et al., Applied the Environmental Microbiology 60 (1994), 126-132). There are however, also known cases in which the overexpression of nonmutated genes is detrimental in a homologous system for the growth of C. glutamicum (for example Eikmanns et al., Microbiology 140 (1994) 1817-1828). In addition, there are significant problems when genes, which do not stem from coryneform bacteria, should be overexpressed in them. In order to express a desired gene in coryneform bacteria, without having to take into consideration a growth blockage by the corresponding gene product, there are various possibilities: a desired gene can be integrated in a single copy number in the chromosome of coryneform bacteria. Since one copy of these genes is provided in the organism, as a rule, no toxic effects arise from the corresponding gene product. A weakness of this process is found in the work-intensive methodology to achieve the desired goal. ln addition, with a single copy number of the inserted gene, seldom is a sufficient quantity of a desired material formed.
An alternative to the integration of a gene in the chromosome of coryneform bacteria is the cloning of a gene on a vector with low copy number in coryneform bacteria. This has the advantage that the corresponding gene product is formed in relatively small amounts and thus usually is not toxic for the cells. However, also in this case a relatively small quantity of the gene product is formed which for biotechnological purposes is a drawback.
It has been desired to form a certain gene product in a large quantity only to a certain point in time in order to overcome the disadvantageous effects of this gene product on the production or growth of the microorganism. To achieve this goal the tendency has been to clone a desired gene without its own promoter behind a controllable promoter. For the controllable Escherichia coli promoters lac, Lambda P.sub.L and trp have already been indicated for use in coryneform bacteria to regulate expression of various genes (Tsuchiya and Morinaga, Bio/Technology 6 (1988) 428-431). However, these promoters have various drawbacks; these promoters do not stem from coryneform organisms and thus have foreign DNA. By the incorporation of such a promoter in coryneform bacteria, these become recombinant organisms for which stricter safety rules apply. In addition, the conditions which are necessary for each of the three promoters for induction of a gene are relatively uninteresting for industrial purposes. Thus the lac-promoter, for induction of a gene, requires the relatively expensive substance IPTG which makes the large-scale use of this promoter uneconomical. The promoter Lambda P.sub.L is activated by heat. Heat, however, not only damages the organisms but also has a detrimental effect to the product formed so that this promoter has no industrial interest for coryneform bacteria. The trp-promoter is activated by a tryptophan deficiency. As a rule coryneform bacteria does not suffer from tryptophan deficiency so that the use of this promoter is preconditioned on the production of coryneform tryptophan deficient mutants. Since the recovery of such mutants is relatively expensive, even the trp-promoter has not found up to now any involvement in the biotechnological use with coryneform bacteria.
The ideal case for a controllable promoter is a coryneform promoter which can be regulated by an easily available inexpensive substance. The hitherto single coryneform promotor described is that of the gene for isocitrate lyase (EP-OS 0 530 765). This promotor leads to expression of genes as long as no sugar is present in the medium. Since sugar is introduced as a carbon source in most fermentation mediums it is sensible to obtain a controllable promoter which also gives rise to expression of a gene in the presence of sugar with an inexpensive inductor.